DEAE-Toyopearl 650M Research Articles

A Consortium of Three Enzymes: Xylanase, Arabinofuranosidase, and Cellulase from Aspergillus sp. which liquefied Coffee Pulp Wastes

Three enzymes released by Aspergillus sp., identified as xylanase, arabinofuranosidase, and cellulase which hydrolyzed coffee pulp during solid state fermentation (SSF) had been investigated. The active fraction of xylanase activity was detected when purification of crude enzymes was performed on DEAE Cellulofine, while cellulase and arabinofuranosidase were fractionated from the previous active fraction using the DEAE Toyopearl 650M and DEAE Mono-Q columns. All enzymes are optimum active at 55°C and stable at temperatures below 50°C and a range of pH 3-6.5. Optimum cellulase activity at pH 5.5 while xylanase and arabinofuranosidase at pH 5. A consortium of these three enzymes can easily hydrolyze the coffee pulp into simple sugar with a degree hydrolysis 79%.

Activity of an A-L-Rhamnosidase Produced by Aspergillus niger During Solid State Fermentation of Coffee Pulp Wastes

An α-L-Rhamnosidase released by Aspergillus niger during solid-state fermentation (SSF) using coffee pulp (CP) wastes media has been investigated. The activity of α-L-Rhamnosidase based on reducing sugar production against 2% CP alkali extract substrate in 50 mM acetate buffer pH 5. The maximum activity of α-L-Rham-nosidase was obtained in sixth-day SSF with reducing sugar pro-duction of 13 μg/mL. The enzyme is actively hydrolyzed 0.1% p-ni-trophenyl-α-L-rhamnopyranoside (PNP-Rha) to 95% from initial concentration. Purification using DEAE-Toyopearl 650M increased hydrolysis activity ten times against the substrate, reaching 134 μg/mL of reducing sugar. Optimum enzyme activity at pH 4.5 and 50°C, while stable at pH and temperature in a pH range of 3.5-7 and below 50°C.

Isolation and Characterization of Water-Soluble Chromoproteins from Arthrospira platensis Cyanobacteria: C-Phycocyanin, Allophycocyanin, and Carotenoid- and Chlorophyll-Binding Proteins

A method using chromatography on DEAE-Toyopearl 650M was developed for the simultaneous extraction of water-soluble chromoproteins from Arthrospira platensis cyanobacteria cells. These chromoproteins were C-phycocyanin, allophycocyanin, and carotenoid- and chlorophyll-binding proteins. The purity of the isolated C-phycocyanin was 4.42 (A620/A280). The allophycocyanin purity was 3.40 (A652/A280). The phycocyanin purity was confirmed by the presence of only two bands obtained during SDS-PAGE: α-subunit (17 kDa) and β-subunit (18 kDa). The isolated carotenoid- and chlorophyll-binding proteins were analyzed by high-performance exclusion chromatography on TSK-GEL 2000SW (XL) with detection at three wavelengths (280, 480, and 678 nm). The spectral, chromatographic, and electrophoretic analyses of chromoproteins, as well as pigment analysis, made it possible to conclude that the carotenoid—chlorophyll a binding protein was a xanthophyll-chlorophyll a protein complex, and the chlorophyll a binding protein was a chlorophyll a protein complex. The molecular weight of the proteins was determined by high-performance exclusion chromatography and SDS-PAGE to be 57 and 16 kDa, respectively. The photoprotective properties of these proteins and their possible functioning as part of evolutionary precursors of photosynthetic systems are discussed.

ВИДІЛЕННЯ, ОЧИЩЕННЯ, СТАБІЛІЗАЦІЯ ТА ХАРАКТЕРИСТИКА ФЛАВОЦИТОХРОМУ b2 ІЗ КЛІТИН НАДПРОДУЦЕНТА OGATAEA POLYMORРHA «tr1» (gcr1 catX CYB2)

A new method of isolation, purification, and stabilization of L-lactate: cytochrome c oxidoreductase (EC 1.1.2.3; flavocytochrome b 2 , FC b 2 ) from the cells of recombinant yeast Ogataea ( Hansenula ) polymorpha “tr1” ( gcr1 catX CYB2 ) has been developed and physico-chemical characterisation of purified preparations has been done. The strain is characterized by impairment of glucose catabolic repression and a five-fold overproduction of FC b 2 , compared to a parent wild-type strain. The effect of several detergents on the yield of membrane-incorporated FC b 2 was studied and the enzyme isolation procedure has been optimized. A new method of affinity chromatography for purification of FC b 2 , based on the use of cytochrome c as a natural ligand, immobilized on aminopropyl silochrome, has been developed. The efficiency of the developed affinity chromatography was compared to ion exchange chromatography on DEAE-Toyopearl 650M cellulose. The specific activity of FC b 2 preparations for both types of chromatography was about 10 U·mg -1 and enzyme yield was in the range of 75–95 %. The purity of the purified FC b 2 was evaluated by SDS-PAAG electrophoresis under denaturing conditions. The effect of a number of stabilizing agents and storage conditions has been investigated in order to provide the maximum activity of the FC b 2 . The best stabilizing effect was observed using 70 % ammonium sulfate and by storage of the preparation at -20 °C. Some physico-chemical parameters of the purified enzyme from O. polymorpha “tr1” cells have been studied (molecular mass and spectral characteristics of oxidized and reduced enzyme forms).

Two-year comparison of the biochemical properties of polyphenol oxidase from Turkish Alyanak apricot (Prunus armenica L.)

Polyphenol oxidase (PPO) was extracted and purified from Turkish Alyanak apricot variety and its characteristics were studied in two consecutive years (2008 and 2009). Three isoenzymes (isoenzyme A1, A2 and B) were obtained upon ammonium sulfate fractionation, ion exchange chromatography using DEAE-Toyopearl 650M and gel filtration chromatography using Sephadex G-100. The isoenzymes exhibited different kinetic properties. Furthermore, year-to-year variability in Km and Vmax values was significant. The pH optimum for enzyme activity was 4.98 for isoenzymes A1 and A2, and 5.8 for isoenzyme B. The isoenzymes A1 and B had optimum temperature at 30°C in both years whereas isoenzyme A2 had maximum activity at 40°C in 2008 and 30°C in 2009. The inactivation kinetics parameters and the effects of inhibitors tested exhibited significant year-to-year variation.

Structure of fructans from three strains of Cronobacter dublinensis

Partially O-acetylated fructan was isolated by phenol—water extraction of cells of Cronobacter dublinensis G2732 followed by anion-exchange chromatography on DEAE-Toyopearl 650M. Structure of the isolated fructan was established by 2D 1H and 13C NMR spectroscopy. The same fructan but lacking O-acetyl groups was isolated from two other C. dublinensis strains, G3947 and G4061. Genes for the fructan biosynthesis were not found at the typical Cronobacter O-antigen gene cluster located between the conserved genes galF and gnd and are evidently located elsewhere on the chromosome.

Suppression of severe lesions, myonecrosis and hemorrhage, caused by Protobothrops flavoviridis venom with its serum proteins

Protobothrops flavoviridis serum proteins precipitated with ammonium sulfate were chromatographed on a DEAE-Toyopearl 650M column at pH 7.5 with stepwise increase or with linear gradient of NaCl concentration. Peaks 3 and 4 serum proteins, obtained by linear gradient elution and named Frde3 and Frde4, contained Habu serum factors (HSF) and phospholipase A2 (PLA2) inhibitors (PfPLI), respectively. The serum proteins eluted at 0.2 M NaCl by stepwise elution, named Fr0.2NaCl, effectively suppressed myonecrosis and hemorrhage caused by P. flavoviridis venom in rat or mouse thigh muscles. The Fr0.2NaCl were fractionated by HPLC and the fractions, after SDS-PAGE, underwent far-western blot analysis with PLA2 ([Asp49]PLA2) and BPI ([Lys49]PLA2) as the probes. Four PfPLIs, namely, PfαPLI-A, PfαPLI-B, PfγPLI-A and PfγPLI-B, were identified together with their selective binding specificities to PLA2 species. In addition, a new 9 kDa protein, which is specifically bound to BPI, was found. Suppression of P. flavoviridis venom-induced severe lesions, such as myonecrosis, hemorrhage and edema, with its serum proteins was histopathologically observed in the present work for the first time. The cooperative use of P. flavoviridis antivenom and its serum proteins as medication for P. flavoviridis snake bites is discussed.

Purification and Partial Characterization of an Acidic α-Glucan–Protein Complex from the Fruiting Body ofPleurotus sajor-cajuand Its Effect on Macrophage Activation

The aim of this study was to purify an acidic α-glucan-protein complex from the fruiting bodies of Pleurotus sajor-caju by using the cell wall-degrading enzymes, xylanase and cellulase. The acidic glucan-protein complex was separated from a polysaccharide extract by using DEAE Toyopearl 650M anion-exchange and Sepharose CL-6B chromatography. Its homogeneity was ensured by high-performance size-exclusion chromatography and agarose gel electrophoresis. The acidic glucan-protein complex had a molecular weight of approximately 182 kDa. Fourier transform infrared spectroscopy of the acidic glucan-protein complex revealed an α-glycosidic bond and the typical characteristics of polysaccharides and proteins. The amino acid composition of the protein moiety was dominated by proline, glycine, glutamic acid and aspartic acid, indicating that the protein was highly flexible and had a negative charge. Atomic force microscopy proved that the acidic α-glucan-protein complex existed in a spherical conformation. The acidic α-glucan-protein complex stimulated the activation of macrophages, including the production of nitric oxide and tumor necrosis factor-α.

Monoterpene Alcohol Metabolism: Identification, Purification, and Characterization of Two Geraniol Dehydrogenase Isoenzymes fromPolygonum minusLeaves

NADP(+)-dependent geraniol dehydrogenase (EC 1.1.1.183) is an enzyme that catalyzes the oxidation of geraniol to geranial. Stable, highly active cell-free extract was obtained from Polygonum minus leaves using polyvinylpolypyrrolidone, Amberlite XAD-4, glycerol, 2-mercaptoethanol, thiourea, and phenylmethylsulfonylfluoride in tricine-NaOH buffer (pH 7.5). The enzyme preparation was separated into two activity peaks, geraniol-DH I and II, by DEAE-Toyopearl 650M column chromatography at pH 7.5. Both isoenzymes were purified to homogeneity in three chromatographic steps. The geraniol-DH isoenzymes were similar in molecular mass, optimal temperature, and pH, but the isoelectric point, substrate specificity, and kinetic parameters were different. The K(m) values for geraniol of geraniol-DH I and II appeared to be 0.4 mM and 0.185 mM respectively. P. minus geraniol-DHs are unusual among geraniol-DHs in view of their thermal stability and optimal temperatures, and also their high specificity for allylic alcohols and NADP(+).

Characterization of α-amylase from mandarin orange honey

Summaryα-Amylase was purified from mandarin orange honey by DEAE-Toyopearl 650M, CM-Toyopearl 650M, and Toyopearl HW-55F column chromatographies, and characterized. The molecular weight of the purified enzyme was estimated to be about 58 kDa by Toyopearl HW-55F gel chromatography and SDS-PAGE, suggesting that the purified enzyme was a monomer. The purified enzyme exhibited optimum activity at pH 5.0 and at 50°C. Inactivation of the enzyme occurred when the pH was lower than 3.0 or higher than 9.0. The enzyme was activated by Co2+, Mn2+, but inhibited by Hg2+, Mg2+, and Fe3+. By TLC analysis, this enzyme was of the α-type that split the interior α-1,4-glycosidic bonds in a random manner. The relative rate of hydrolysis of the polymeric substrate decreased with decreasing percentage of α-1,4-linkages and with increasing percentage of α-1,6-linkages in substrate similar to the results from commercially available honey.

Purification, characterization and thermal inactivation kinetics of a non-regioselective thermostable lipase from a genotypically identified extremophilic Bacillus subtilis NS 8

Thermostable lipase produced by a genotypically identified extremophilic Bacillus subtilis NS 8 was purified 500-fold to homogeneity with a recovery of 16% by ultrafiltration, DEAE-Toyopearl 650M and Sephadex G-75 column. The purified enzyme showed a prominent single band with a molecular weight of 45 kDa. The optimum pH and temperature for activity of lipase were 7.0 and 60°C, respectively. The enzyme was stable in the pH range between 7.0 and 9.0 and temperature range between 40 and 70°C. It showed high stability with half-lives of 273.38 min at 60°C, 51.04 min at 70°C and 41.58 min at 80°C. The D-values at 60, 70 and 80°C were 788.70, 169.59 and 138.15 min, respectively. The enzyme's enthalpy, entropy and Gibb's free energy were in the range of 70.07-70.40 kJ mol(-1), -83.58 to -77.32 kJ mol(-1)K(-1) and 95.60-98.96 kJ mol(-1), respectively. Lipase activity was slightly enhanced when treated with Mg(2+) but there was no significant enhancement or inhibition of the activity with Ca(2+). However, other metal ions markedly inhibited its activity. Of all the natural vegetable oils tested, it had slightly higher hydrolytic activity on soybean oil compared to other oils. On TLC plate, the enzyme showed non-regioselective activity for triolein hydrolysis.

Partial purification and characterization of polyphenol oxidase from sugarcane (Saccharum officinarum L.)

Polyphenol oxidase (PPO) of sugarcane was extracted by using 0.02 mol/L phosphate buffer at pH = 6.8 containing 1.5% polyvinylpolypyrrolidone and 0.5% Triton X-100, and then partially purified by 80% ammonium sulfate fractionation, dialysis, and column chromatography on DEAE-Toyopearl 650M, Sephadex G-100. PPO activity was purified 37.6-fold with a recovery of 18.4%. The PPO showed activity to catechol, chlorogenic acid, 4-methylcatechol, caffeic acid and ferulic acid, but not to l-tyrosine. Optimum conditions (pH value and temperature) for PPO were determined using the five substances. PPO activity is quite thermostable between 20 and 30 °C. After heating for 10 min at 80 °C 90% of the activity is lost. Km and Vmax values of PPO were calculated for each substrate and the best substrate of PPO was chlorogenic acid. PPO was markedly inhibited by metal ions (Cu2+, Al3+, and Mg2+) at 1 and 10 mmol/L, and strongly inhibited by NaHSO3 and ascorbic acid at 1 mmol/L.

Characterization of a Lipase from Sporidiobolus pararoseus 25-A Which Produces Cheese Flavor

A lipase from a Sporidiobolus pararoseus strain was purified from culture filtrate by (NH4)2SO4 precipitation, and DEAE-Toyopearl 650M, Butyl-Toyopearl 650M, and Toyopearl HW-55 chromatography. The purified enzyme appeared as a single band with a molecular mass of 37 kDa by SDS-PAGE. The optimum temperature and pH were approximately 60°C and 6.0, respectively. The specificity toward triglyceride was similar to that of pregastric esterase Lipase PGET. Addition of the lipase during the mozzarella cheese-making process produced a strong cheese flavor. Taken together, the lipase produced by S. pararoseus is considered a potential replacement of pregastric esterase in the dairy industry.

Removal and inactivation of viruses during the manufacture of a high-purity antihemophilic factor IX from human plasma

The purpose of this study was to evaluate the efficacy and mechanisms of the solvent/detergent (S/D) treatment, DEAE-toyopearl 650M anion-exchange column chromatography, heparin-sepharose 6FF affinity column chromatography, and Viresolve NFP filtration steps employed in the manufacture of high-purity antihemophilic factor IX (Green-Nine VF) from human plasma, with regard to removal and/or inactivation of blood-borne viruses. A variety of experimental model viruses for human pathogenic viruses, including human immunodeficiency virus (HIV), bovine herpes virus (BHV), bovine viral diarrhoea virus (BVDV), hepatitis A virus (HAV), murine encephalomyocarditis virus (EMCV), and porcine parvovirus (PPV), were all selected for this study. Samples from relevant stages of the production process were spiked with each virus and subjected to scale-down processes mimicking the manufacture of high-purity factor IX. Samples were collected at each step, immediately titrated using a 50% tissue culture infectious dose (TCID50), and virus reduction factors were evaluated. S/D treatment using the organic solvent, tri (n-butyl) phosphate (TNBP), and the detergent, Tween 80, was a robust and effective step in inactivation of enveloped viruses. Titers of HIV, BHV, and BVDV were reduced from the initial titer of 6.06, 7.72, and 6.92 log10 TCID50, respectively, reaching undetectable levels within 1 min of S/D treatment. DEAE-toyopearl 650M anion-exchange column chromatography was found to be a moderately effective step in the removal of HAV, EMCV, and PPV with log reduction factors of 1.12, 2.67, and 1.38, respectively. Heparin-sepharose 6FF affinity column chromatography was also moderately effective for partitioning BHV, BVDV, HAV, EMCV, and PPV with log reduction factors of 1.55, 1.35, 1.08, 1.19, and 1.61, respectively. The Viresolve NFP filtration step was a robust and effective step in removing all viruses tested, since HIV, BHV, BVDV, HAV, EMCV, and PPV were completely removed during the filtration step with log reduction factors of ≥ 5.51, ≥ 5.76, ≥ 5.18, ≥ 5.34, ≥ 6.13, and ≥ 4.28, respectively. Cumulative log reduction factors of HIV, BHV, BVDV, HAV, EMCV, and PPV were ≥ 10.52, ≥ 12.07, ≥ 10.49, ≥ 7.54, ≥ 9.99, and ≥ 7.24, respectively. These results indicate that the production process for GreenNine VF has a sufficient virus reduction capacity for achievement of a high margin of virus safety.

Alpha-Amylase from Persimmon Honey: Purification and Characterization

The α-amylase was extracted from pure persimmon honey and purified by DEAE-Toyopearl 650M, CM-Toyopearl 650M, and Toyopearl HW-55F column chromatographies. Molecular weight of purified enzyme was estimated to be about 58 kDa by Toyopearl HW-55F gel chromatography and SDS-PAGE, respectively suggested that the purified enzyme was a monomer. Optimum pH of the enzyme was 6.0−7.0 and optimum temperature 40°C. The enzyme was extremely inactivated at pH was higher than 7.0 or lower than 5.0. Heat inactivation occurred at 40°C. This enzyme activated by Ca 2+ , Mn2+, PCMB, and DTNB, but inhibited by Ba2+, Fe3+, Hg2+, Mg2+, and iodoacetic acid. The purified enzyme was of α -type by TLC analysis. The relative rate of hydrolysis of the polymeric substance decreased with decreasing percentage of α -1,4-linkages and with increasing percentage of α -1,6-linkages in substrate similar to the results from commercially available honey.

Purification, Characterization and Application of .ALPHA.-Amylase from Pseudozyma aphidis I-8

A yeast strain I-8 was isolated as an α-amylase producer from digestive juice of Nepenthes bicalarate. The yeast was identified as Pseudozyma aphidis by the morphological test and comparative 26S rDNA-D1/D2 and ITS-5.8S rDNA gene sequence analysis. The α-amylase was purified from the culture filtrate by (NH4)2SO4 precipitation, DEAE-TOYOPEARL 650M, Butyl-TOYOPEARL 650M, Hydroxylapatite and TOYOPEARL HW-55 chromatography. The purified enzyme was shown as a single band and the molecular mass was 55 kDa by SDS-PAGE. The specific activity was 1679 U/mg protein. The optimum temperature and pH were around 60°C and 5.0, respectively. The enzyme was stable in a pH range from 5.0 to 9.0 and at below 60°C. The enzyme hydrolyzed soluble starch and released glucose, maltose and oligosaccharides. Maltooligosaccharides (G3-G5) were also favorable substrate but it showed no activity toward maltose, isomaltose or pullulan. On the hydrolysis of soluble starch, the iodine color of the reaction mixture disappeared at almost 10% of reducing sugar formation and the hydrolysis limit was about 70% of soluble starch. From these results, the α-amylase was recognized as a unique α-amylase. The α-amylase was applied to the bread making process. Addition of the α-amylase to the bread making process presented no effect toward the crumb softness or color but the improved taste of the bread by sensory evaluation.

Purification and Partial Characterization of Major Viscous Protein from Yam (Dioscorea opposita Thunb.) Tuber Mucilage tororo

Viscous protein was purified from yam (Dioscorea opposita Thunb.) tuber mucilage tororo by Phenyl-Toyopearl 650M, DEAE-Toyopearl 650M, and CM-Toyopearl 650M column chromatographies, and partially characterized. The molecular weight of the purified protein was estimated to be about 200 kDa, by Native-PAGE and Toyopearl HW-55F gel filtration. Moreover, it had identical subunits of molecular weight of about 32 kDa, either without or with 2-mercaptoethanol, respectively. This protein was identified as dioscorin, the major storage proteins in yam species tubers, when comparing the sequence obtained in an automatic Edman sequencer with the database using FASTA WWW System. On the other hand, the purified protein showed carbonic anhydrase activity and the activity was not inhibited by 2,6-pyridinedicarboxylic acid: zinc atoms were absent in the purified protein. The optimum pH in viscosity was about 5.8 and optimum temperature 20–30°C. Heat denaturation of the protein occurred at about 40°C, but the protein retained more than 66 % of the original viscosity at 70°C for 30 min.

Extracellular Oxidases of the Lignin-Degrading Fungus Panus tigrinus

Two extracellular oxidases (laccases) were isolated from the extracellular fluid of the fungus Panus (Lentinus) tigrinus cultivated in low-nitrogen medium supplemented with birch sawdust. The enzymes were purified by successive chromatography on columns with TEAE-cellulose and DEAE-Toyopearl 650M. Both oxidases catalyze oxidation of pyrocatechol and ABTS. Moreover, oxidase 1 also catalyzes oxidation of guaiacol, o-phenylenediamine, and syringaldazine. The enzymes have identical pH (7.0) and temperature (60-65 degrees C) optimums. Absorption spectra of the oxidases differ from the spectra of typical "blue" laccases and are similar to the spectrum of yellow oxidase.

Aeromonas sp. MN44의 특성과 망간 산화에 관한 연구

충청남도 목천과 충청북도 오창 근교의 토양으로부터 망간을 산화하는 64 집락을 분리하고 이 중 망간 산화능이 가장 우수한 한 균주를 최종 선별하여 생리, 생화학적 특성을 조사하고, 16S rRNA 염기 서열분석 등을 통하여 동정한 결과 최종 선별된 균주는 Aeromonas sp. MN44로 확인되었다. 최종 선별된 Aeromenas sp. MN44는 lactose를 제외한 여러 당들은 이용하지 못하였으며, 중금속내성은 lithium과 manganese에 대해서는 mg/ml 단위의 높은 농도까지 중금속 내성을 가지고 있었지만 cadmium에는 전혀 내성을 나타내지 않았다. 또 kanamycin, chloramphenicol, ampicillin, tetracycline, spectinomycin등 조사한 모든 항생제에 대해 전혀 내성을 갖지 않았다. Aeromonas sp. MN44가 생성하는 망간산화물질의 최적 pH는 pH 7.4로 확인되었으며, 이 균이 생성하는 망간 산화 factor는 proteinase K와 가열처리에 의해 저해되는 단백질이고, ammonium sulfate 침전과 ion exchange chromatography 그리고 gel filtration의 단계를 통해 부분 정제한 망간 산화 factor의 분자량은 약 113 kDa로 확인되었다. Sixty four bacterial colonies which were able to oxidize the manganese were isolated from soil samples in Mokcheon and Ochang area. Among them, one bacterial strain was selected for this study based on its higher manganese oxidation, and this selected bacterial strain was identified as Aeromonas sp. MN44 through physiological-biochemical test and analysis of its 16s rRNA sequence. Aeromonas sp. MN44 was able to utilize lactose but did not utilize various carbohydrates as a sole carbon source. Aeromonas sp. MN44 showed a very sensitive to antibiotics such as kanamycin, chloramphenicol, ampicillin, tetracycline and spectinomycin, and heavy metal such as cadmium. But this strain showed a high resistance up to mg/ml unit to heavy metals such as lithium and manganese. Optimal manganese oxidation condition of Aeromonas sp. MN44 was pH 7.4 and manganese oxidation activity was inhibited by proteinase K and boiling treatment. So, we concluded that this factor was protein. The manganese oxidizing factor produced by Aeromonas sp. MN44 was partial purified by ammonium sulfate precipitation, DEAE-Toyopearl 650M ion exchange chromatography and Sephadex gel filtration chromatography. Its molecular mass was about 113 kDa.

Purification, Characterization, and a Potential Application of .BETA.-Glucosidase from Aspergillus pulverulentus YM-80

A fungus, strain YM-80, isolated from a soil sample produced β-glucosidase ( β-D-glucoside glucohydrolase EC 3.2.1.21) in solid culture. The fungus was identified as Aspergillus pulverulentus from its taxonomical characteristics. The β-glucosidase was purified 18-fold from the culture extract by (NH4)2SO4 precipitation, DEAE-Toyopearl 650M, Butyl-Toyopearl 650M and Sephacryl S-200 chromatography. The purified enzyme appeared as a single band on SDS-PAGE. The molecular weight of the purified enzyme was estimated to be 118,000 by SDS-PAGE and 240,000 by gel filtration. The enzyme was an acidic protein with a pI of 4.5 and the specific activity was 308 U/mg protein. The optimum temperature and pH for the enzyme activity were 60°C and 4.0, respectively. This enzyme was stable in the pH range 3.0-7.0 and also up to 50°C. The activity of the enzyme was completely inhibited by Hg2+ and SDS. The purified enzyme was active with a broad range of β-linked glucosides but not α-linked saccharides. Added during tofu processing, the enzyme was useful for the preparation of aglycon isoflavone-enriched tofu by hydrolysis of the glucoside form. However, isoflavones with 6"-O-acetyl and 6"-O-malonyl glucosides were not converted into the aglycon form.